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Biomedical subjects

Michael K Rosen

Publications and source records attributed to Michael K Rosen.

At least 19 recordsLinked to original sources
PubMed2025

An electrostatic repulsion model of centromere organisation.

During cell division, chromosomes reorganise into compact bodies in which centromeres localise precisely at the chromatin surface1-4 to enable kinetochore-microtubule interactions essential for genome segregation5-8. The physical principles guiding this centromere positioning remain unknown. Here, we reveal that human core centromeres are directed to the chromatin surface by repulsion of centromere-associated proteins - independent of condensin-mediated loop extrusion and microtubule engagement. Using cellular perturbations, biochemical reconstitution, and multiscale molecular dynamics simulations, we show that chromatin surface localisation emerges from repulsion between condensed chromatin and both the kinetochore and the highly negatively charged centromere protein, CENP-B. Together, these elements form a centromeric region composed of two domains with opposing affinities, one favouring integration within the mitotic chromosome and the other favouring exposure to the surrounding cytoplasm, thereby driving surface positioning. Tethering synthetic negatively charged proteins to chromatin was sufficient to recapitulate this surface localisation in cells and in vitro, indicating that electrostatic repulsion is a key determinant of surface localisation. These findings demonstrate that centromere layering is not hardwired by chromatin folding patterns but instead emerges from phase separation in chromatin. Our work uncovers electrostatic polarity as a general and programmable mechanism to spatially organise chromatin.

Journal Article↗
PubMed2006

Signal transduction pathway of TonB-dependent transporters.

Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.

Bacterial Proteins↗
PubMed2006

Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLalpha and mDia1.

Diaphanous-related formins (DRFs) are key regulators of actin cytoskeletal dynamics whose in vitro actin assembly activities are thought to be regulated by autoinhibition. However, the in vivo consequences of autoinhibition and the involvement of DRFs in specific biological processes are not well understood. In this study, we show that in the DRFs FRLalpha (formin-related gene in leukocytes alpha) and mouse diaphanous 1, autoinhibition regulates a novel membrane localization activity in vivo as well as actin assembly activity in vitro. In FRLalpha, the Rho family guanosine triphosphatase Cdc42 relieves the autoinhibition of both membrane localization and biochemical actin assembly activities. FRLalpha is required for efficient Fc-gamma receptor-mediated phagocytosis and is recruited to the phagocytic cup by Cdc42. These results suggest that mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate a novel role for formins in immune function.

Actins↗
PubMed2006

Specific patterns of Cdc42 activity are related to distinct elements of T cell polarization.

T cell polarization toward and within the cellular interface with an APC is critical for effective T cell activation. The Rho family GTPase Cdc42 is a central regulator of cellular polarization. Using live-cell imaging, we characterized the spatiotemporal patterns of Cdc42 activity and their physiological regulation. Using three independent means of experimental manipulation of Cdc42 activity, we established that Cdc42 is a critical regulator of T cell actin dynamics, TCR clustering, and cell cycle entry. Using quantification of three-dimensional data, we could relate distinct spatiotemporal patterns of Cdc42 activity to specific elements of T cell activation. This result suggests that Cdc42 activity in specific locations at specific times is most critical for its function in T cell activation.

Actins↗
PubMed2006

Biochemical properties and inhibitors of (N-)WASP.

The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho GTPase Cdc42 and a key component of signaling pathways that regulate the actin cytoskeleton. WASP is regulated by a number of ligands, and the mechanisms by which these act are beginning to be understood through detailed biochemical analyses. Here we describe the protocols we use to study WASP proteins, including the methods we use to purify signaling components and the assays we use to quantitatively characterize the biochemical and biophysical properties of WASP, its activation by Cdc42, and its inhibition by the small molecule wiskostatin. These methods have broad use within the WASP-related cytoskeletal-signaling pathway but are also applicable to investigations of other intramolecular and intermolecular interactions.

Actin-Related Protein 2-3 Complex↗
PubMed2005

NMR analyses of the activation of the Arp2/3 complex by neuronal Wiskott-Aldrich syndrome protein.

The VCA domain of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a potent activator of the Arp2/3 complex, a 240 kDa heteroheptameric actin-nucleating assembly. We used site-directed spin labeling of N-WASP peptides in conjunction with methyl-TROSY spectra of the intact, selectively labeled Arp2/3 complex to identify regions of the VCA that are proximal to the ARPC3 subunit of the assembly. We also cross-linked CA peptides to the Arp3, Arp2, ARPC1, and ARPC3 subunits. The combined data suggest that the extreme C-terminus of the A region and the C-terminus of the C region of N-WASP are proximal to ARPC3. These results have implications for the mechanism of Arp2/3 complex activation by VCA peptides. This study also demonstrates the utility of NMR spectroscopy for studying ligand binding events in large, asymmetric, macromolecular assemblies.

Actin-Related Protein 2-3 Complex↗
PubMed2005

Acidic region tyrosines provide access points for allosteric activation of the autoinhibited Vav1 Dbl homology domain.

Autoinhibited proteins serve key roles in many signal transduction pathways, and therefore proper regulation of these proteins is critical for normal cellular function. Proto-oncogene Vav1 is an autoinhibited guanine nucleotide exchange factor (GEF) for Rho family GTPases. The core autoinhibitory module of Vav1 consists of the catalytic Dbl homology (DH) domain bound through its active site to an alpha helix centered about Tyr174 in the Acidic (Ac) region of the protein. Phosphorylation of Tyr174 and two other tyrosines in the Ac region, Tyr142 and Tyr160, relieves autoinhibition and activates the catalytic DH domain. In this study, we use biochemical and structural analyses of the Vav1 Ac and DH domains to examine the kinetic and thermodynamic properties of Vav1 activation by the Src family kinase, Lck, and the role of the Lck SH2 domain in this process. We find that in the Ac-DH fragment of Vav1, Tyr174, but not Tyr142 or Tyr160, is protected from phosphorylation by interactions with the DH domain. Binding of the Lck SH2 domain to phosphorylated Tyr142 increases kcat/KM for Tyr174 by 4-fold, likely because the kinase domain can act on the substrate effectively in an intramolecular fashion. These studies of the autoinhibited Ac-DH module provide the foundation for a quantitative structural and thermodynamic understanding of the regulation of full length Vav1. Moreover, kinetic pathways involving initial interactions with exposed sites or "access points", as observed here for Vav1, may be generally important in the regulation of many autoinhibited proteins.

Allosteric Regulation↗
PubMed2005

Protein-tyrosine kinase and GTPase signals cooperate to phosphorylate and activate Wiskott-Aldrich syndrome protein (WASP)/neuronal WASP.

Protein-tyrosine kinases and Rho GTPases regulate many cellular processes, including the reorganization and dynamics of the actin cytoskeleton. The Wiskott-Aldrich syndrome protein (WASP) and its homolog neuronal WASP (N-WASP) are effectors of the Rho GTPase Cdc42 and provide a direct link between activated membrane receptors and the actin cytoskeleton. WASP and N-WASP are also regulated by a large number of other activators, including protein-tyrosine kinases, phosphoinositides, and Src homology 3-containing adaptor proteins, and can therefore serve as signal integrators inside cells. Here we show that Cdc42 and the Src family kinase Lck cooperate at two levels to enhance WASP activation. First, autoinhibition in N-WASP decreases the efficiency (kcat/Km) of phosphorylation and dephosphorylation of the GTPase binding domain by 30- and 40-fold, respectively, and this effect is largely reversed by Cdc42. Second, Cdc42 and the Src homology 3-Src homology 2 module of Lck cooperatively stimulate the activity of phosphorylated WASP, with coupling energy of approximately 2.4 kcal/mol between the two activators. These combined effects provide mechanisms for high specificity in WASP activation by coincident GTPase and kinase signals.

Actins↗
PubMed2005

A novel mechanism of actin filament processive capping by formin: solution of the rotation paradox.

The FH2 domains of formin family proteins act as processive cappers of actin filaments. Previously suggested stair-stepping mechanisms of processive capping imply that a formin cap rotates persistently in one direction with respect to the filament. This challenges the formin-mediated mechanism of intracellular cable formation. We suggest a novel scenario of processive capping that is driven by developing and relaxing torsion elastic stresses. Based on the recently discovered crystal structure of an FH2-actin complex, we propose a second mode of processive capping-the screw mode. Within the screw mode, the formin dimer rotates with respect to the actin filament in the direction opposite to that generated by the stair-stepping mode so that a combination of the two modes prevents persistent torsion strain accumulation. We determine an optimal regime of processive capping, whose essence is a periodic switch between the stair-stepping and screw modes. In this regime, elastic energy does not exceed feasible values, and supercoiling of actin filaments is prevented.

Actin Cytoskeleton↗
PubMed2005

Structural basis of Rho GTPase-mediated activation of the formin mDia1.

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. We report the crystal structure of the dimeric regulatory domain of the DRF, mDia1. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design.

Animals↗
PubMed2005

The nucleotide switch in Cdc42 modulates coupling between the GTPase-binding and allosteric equilibria of Wiskott-Aldrich syndrome protein.

The GTP/GDP nucleotide switch in Ras superfamily GTPases generally involves differential affinity toward downstream effectors, with the GTP-bound state having a higher affinity for effector than the GDP-bound state. We have developed a quantitative model of allosteric regulation of the Wiskott-Aldrich syndrome protein (WASP) by the Rho GTPase Cdc42 to better understand how GTPase binding is coupled to effector activation. The model accurately predicts WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 as functions of a two-state allosteric equilibrium in WASP. The ratio of GTPase affinities for the inactive and active states of WASP is appreciably larger for Cdc42-GTP than for Cdc42-GDP. The greater ability to distinguish between the two states of WASP makes Cdc42-GTP a full WASP agonist, whereas Cdc42-GDP is only a partial agonist. Thus, the nucleotide switch controls not only the affinity of Cdc42 for its effector but also the efficiency of coupling between the Cdc42-binding and allosteric equilibria in WASP. This effect can ensure high fidelity and specificity in Cdc42 signaling in crowded membrane environments.

Actin-Related Protein 2↗
PubMed2005

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain.

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

Actin Cytoskeleton↗
PubMed2004

Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation.

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors because they also occur in noncatalytic proteins and because allosteric inhibitors may avoid specificity problems inherent in active site-directed inhibitors. We report here the identification of wiskostatin, a chemical inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP). Wiskostatin interacts with a cleft in the regulatory GTPase-binding domain (GBD) of WASP in the solution structure of the complex. Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state. The use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even in cases where such sites have not been naturally evolved in a target.

Allosteric Site↗
PubMed2004

A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting.

Remodeling of the actin cytoskeleton is controlled by signaling pathways that include the Wiskott-Aldrich syndrome protein (WASP). WASP is regulated by autoinhibition, and the intramolecular contacts that inactivate the protein can be relieved through binding to the Rho-family GTPase Cdc42. Here, we show that the allosteric regulation of WASP can be quantitatively described by a two-state equilibrium between an active, largely unfolded conformation that is able to stimulate the Arp2/3 complex, and an inactive, folded conformation. The model is parameterized by the stability of WASP against unfolding and by the Cdc42 affinities of WASP constructs that mimic the unfolded and folded conformations. The model is consistent with NMR spectra of GTPase-bound WASP, and accurately predicts changes of amide hydrogen exchange behavior and Cdc42 affinity as a function of WASP stability. The results provide a thermodynamic rationale for the GTPase-independent recruitment of WASP and other autoinhibited effectors to their sites of activity. They also explain how basal activity is suppressed and confirm that WASP needs to integrate multiple cooperative inputs for maximal activation. Our analysis suggests that, in general, simple modulation of a two-state equilibrium may determine several regulatory functions, allowing the generation of complex signaling behavior in vivo.

Allosteric Regulation↗
PubMed2003

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP.

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.

Actins↗
PubMed2003

NMR analysis of methyl groups at 100-500 kDa: model systems and Arp2/3 complex.

Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form. Here, we present (1)H/(13)C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to approximately 560 kDa. Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to approximately 330 ns. We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained (1)H/(13)C-HSQC spectra of this assembly. Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 microM. Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies.

Actin-Related Protein 2↗
PubMed2003

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling.

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.

Energy Transfer↗